A large number of applications involving the magnetic separation of biological cells are described in the literature, for example in U.S. Pat. No. 4,710,472 and the many publications cited therein. Many such applications require not only the separation of one or more specific types of cells (hereinafter called “target cells”), but also the maintenance of the quality of the cell membranes in the target cells and/or in the untargeted cells. Thus, in a positive selection process, the target cells are separated from a sample for examination or use for research, diagnostic or clinical purposes; whereas in a depletion process, the sample is depleted of the target cells for examination or use of the untargeted cells. The separation of target cells from the untargeted cells, and the maintenance of the membranes of both the target cells and untargeted cells, are particularly important in research presently being conducted with lymphocyte populations and their role in the early detection of cancer.
One technique in present use for the separation of biological cells utilizes the MiniMACS Separation Columns (Miltenyi Biotec GmbH). This technique uses paramagnetic microbeads which are extremely small, about 50 nm in diameter, i.e., about one million times smaller in volume than that of eukaryotic cells, compared to the size of a virus. Such magnetic microbeads are produced with selective affinities for certain cells, i.e., the target cells, such that they magnetically label or stain the target cells. The sample is introduced into a magnetic separation column including a liquid-pervious magnetic body, e.g., steel wool or mesh, and a magnetic field is applied across the column such that the magnetically stained cells are retained in the liquid-pervious magnetic body of the column, while the unstained cells pass through the column. In this known process, however, it was found that the membranes of the cells are excessively damaged by the liquid-pervious magnetic body, which reduces the effectiveness of the technique for research or clinical purposes.
In some cases the target cells are rare in a sample, such as cancer cell, stem cells or fetal cells posing a challenge for the separation process. As a non-limiting example fetal cells are referred below.
The presence of fetal cells in maternal blood has been known for over a century since Schmorl (1) demonstrated trophoblast cells in the pulmonary circulation of women who died of eclampsia. Since then, five different fetal cell types have been demonstrated to circulate in maternal blood, including trophoblast cells (1), lymphocytes (2), nucleated erythrocytes (NRBCs) (3), granulocytes (4), and stem cells (5). The frequency of fetal cells in maternal circulation is very low, with an average of 1.2 cells/ml maternal blood as detected by a quantitative PCR technique (6). Among all of the fetal cell types isolated from maternal blood, NRBCs have emerged as the best candidate cells to be used in noninvasive prenatal diagnosis (7).
Researchers have attempted separation and enrichment of fetal NRBC using fluorescence activated cell sorting (FACS) and magnetic cell sorting (MACS) methods, including a large scale study funded by the NIH (NIFTY study). This study achieved low sensitivity of fetal cell detection with 13% recovery for FACS and 44% for MACS (the leading magnetic separation technology) (8) and concluded that as the proportion of fetal NRBC in maternal blood is low, and current separation techniques have low recovery rates, the utilization of FNRBC is not feasible for clinical application. As it is extremely difficult to separate these cells, alternative and less effective methods based on different genetic materials are explored, such as DNA fragments (9). These materials must be multiplied using PCR in order to effectively analyze them and cannot provide the same level of conclusive results regarding trisomies as FNRBCs might.
U.S. Pat. No. 6,482,328 to some of the inventors of the present invention and others discloses a method and apparatus for magnetically separating target particles of a selected type from a sample in order to produce concentration of the target particles in the sample, or depletion of the sample with respect to the target particles, by producing a sample mixture of the sample with magnetic particles having a selective affinity to magnetically stain the target particles.
There remains an unmet need for rapid and reliable magnetic separation of living rare cells with high yield and high purity.